In the expanding arena of drug discovery and combinatorial chemistry to generate candidate compounds, it would be very useful to be able to rapidly screen a large number of substances, via a high throughput screen, for their physiological impact on animals and humans. Before testing the efficacy of a “partially qualified” drug candidate on animals, the drug could first be screened for its biological activity and potential toxicity with living cells. The physiological response to the drug candidate could then be anticipated from the results of these cell screens.
Traditionally, “lead compounds” have moved quickly to extensive animal studies that are both time-consuming and costly. Furthermore, extensive drug testing in animals is becoming less culturally acceptable. Screening drug candidates according to their interaction with living cells, prior to animal studies, can reduce the number of animals required in subsequent drug screening processes by eliminating some drug candidates before going to animal trials. However, manipulation and analysis of drug-cell interactions using current methods does not allow for both high throughput and high biological content screening, due to the small number of cells and compounds that can be analyzed in a given period of time, the cumbersome methods required for compound delivery, and the large volumes of compounds required for testing.
High throughput screening of nucleic acids and polypeptides has been achieved using DNA chip technologies. In typical DNA analysis methods, DNA sequences of 10 to 14 nucleotides are attached in defined locations (or spots), up to tens of thousands in number, on a small glass plate. (U.S. Pat. No. 5,556,752, hereby incorporated by reference). This creates an array of spots of DNA on a given glass plate. The location of a spot in the array provides an address for later reference to each spot of DNA. The DNA sequences are then hybridized with complementary DNA sequences labeled with fluorescent molecules. Signals from each address on the array are detected when the fluorescent molecules attached to the hybridizing nucleic acid sequences fluoresce in the presence of light. These devices have been used to provide high throughput screening of DNA sequences in drug discovery efforts and in the human genome sequencing project. Similarly, protein sequences of varying amino acid lengths have been attached in discrete spots as an array on a glass plate. (U.S. Pat. No. 5,143,854, incorporated by reference herein).
The information provided by an array of either nucleic acids or amino acids bound to glass plates is limited according to their underlying “languages”. For example, DNA sequences have a language of only four nucleic acids and proteins have a language of about 20 amino acids. In contrast, a living cell, which comprises a complex organization of biological components, has a vast “language” with a concomitant multitude of potential interactions with a variety of substances, such as DNA, RNA, cell surface proteins, intracellular proteins and the like. Because a typical target for drug action is with and within the cells of the body, cells themselves provide an extremely useful screening tool in drug discovery when combined with sensitive detection reagents. It thus would be most useful to have high throughput, high content screening devices to provide high content spatial information at the cellular and subcellular level as well as temporal information about changes in physiological, biochemical and molecular activities.
Microarrays of Cells
Methods have been described for making micro-arrays of a single cell type on a common substrate for other applications. One example of such methods is photochemical resist-photolithograpy (Mrksich and Whitesides, Ann. Rev. Biophys. Biomol. Struct. 25:55-78, 1996), in which a glass plate is uniformly coated with a photoresist and a photo mask is placed over the photoresist coating to define the “array” or pattern desired. Upon exposure to light, the photoresist in the unmasked areas is removed. The entire photolithographically defined surface is uniformly coated with a hydrophobic substance, such as an organosilane, that binds both to the areas of exposed glass and the areas covered with the photoresist. The photoresist is then stripped from the glass surface, exposing an array of spots of exposed glass. The glass plate then is washed with an organosilane having terminal hydrophilic groups or chemically reactable groups such as amino groups. The hydrophilic organosilane binds to the spots of exposed glass with the resulting glass plate having an array of hydrophilic or reactable spots (located in the areas of the original photoresist) across a hydrophobic surface. The array of spots of hydrophilic groups provides a substrate for non-specific and non-covalent binding of certain cells, including those of neuronal origin (Kleinfeld et al., J. Neurosci. 8:4098-4120, 1988).
In another method based on specific yet non-covalent interactions, stamping is used to produce a gold surface coated with protein adsorptive alkanethiol. (U.S. Pat. No. 5,776,748; Singhvi et al., Science 264:696-698, 1994). The bare gold surface is then coated with polyethylene-glycol-terminated alkanethiols that resist protein adsorption. After exposure of the entire surface to laminin, a cell-binding protein found in the extracellular matrix, living hepatocytes attach uniformly to, and grow upon, the laminin coated islands (Singhvi et al. 1994). An elaboration involving strong, but non-covalent, metal chelation has been used to coat gold surfaces with patterns of specific proteins (Sigal et al., Anal. Chem. 68:490-497, 1996). In this case, the gold surface is patterned with alkanethiols terminated with nitriloacetic acid. Bare regions of gold are coated with tri(ethyleneglycol) to reduce protein adsorption. After adding Ni2+, the specific adsorption of five histidine-tagged proteins is found to be kinetically stable.
More specific single cell-type binding can be achieved by chemically crosslinking specific molecules, such as proteins, to reactable sites on the patterned substrate. (Aplin and Hughes, Analyt. Biochem. 113:144-148, 1981). Another elaboration of substrate patterning optically creates an array of reactable spots. A glass plate is washed with an organosilane that chemisorbs to the glass to coat the glass. The organosilane coating is irradiated by deep UV light through an optical mask that defines a pattern of an array. The irradiation cleaves the Si—C bond to form a reactive Si radical. Reaction with water causes the Si radicals to form polar silanol groups. The polar silanol groups constitute spots on the array and are further modified to couple other reactable molecules to the spots, as disclosed in U.S. Pat. No. 5,324,591, incorporated by reference herein. For example, a silane containing a biologically functional group such as a free amino moiety can be reacted with the silanol groups. The free amino groups can then be used as sites of covalent attachment for biomolecules such as proteins, nucleic acids, carbohydrates, and lipids. The non-patterned covalent attachment of a lectin, known to interact with the surface of cells, to a glass substrate through reactive amino groups has been demonstrated (Aplin & Hughes, 1981). The optical method of forming a micro-array of a single cell type on a support requires fewer steps and is faster than the photoresist method, (i.e., only two steps), but it requires the use of high intensity ultraviolet light from an expensive light source.
In all of these methods, the result is a micro-array of a single cell type, since the biochemically specific molecules are bound to the micro-patterned chemical array uniformly. In the photoresist method, cells bind to the array of hydrophilic spots and/or specific molecules attached to the spots which, in turn, bind cells. Thus cells bind to all spots in the array in the same manner. In the optical method, cells bind to the array of spots of free amino groups by adhesion. There is little or no differentiation between the spots of free amino groups. Again, cells adhere to all spots in the same manner, and thus only a single type of cell interaction can be studied with these cell arrays because each spot on the array is essentially the same as another. Such cell arrays are inflexible in their utility as tools for studying a specific variety of cells in a single sample or a specific variety of cell interactions. Thus, there exists a need for arrays of multiple cell types on a common substrate, in order to increase the number of cell types and specific cell interactions that can be analyzed simultaneously, as well as methods of producing these micro-arrays of multiple cell types on a common substrate, in order to provide for high throughput and high biological content screening of cells.
Optical Reading of Cell Physiology
Performing a high throughput screen on many thousands of compounds requires parallel handling and processing of many compounds and assay component reagents. Standard high throughput screens use homogeneous mixtures of compounds and biological reagents along with some indicator compound, loaded into arrays of wells in standard microplates with 96 or 384 wells. (Kahl et al., J. Biomol. Scr. 2:33-40, 1997). The signal measured from each well, either fluorescence emission, optical density, or radioactivity, integrates the signal from all the material in the well giving an overall population average of all the molecules in the well. This type of assay is commonly referred to as a homogeneous assay.
U.S. Pat. No. 5,581,487 describes an imaging plate reader that uses a CCD detector (charge-coupled optical detector) to image the whole area of a 96 well plate. The image is analyzed to calculate the total fluorescence per well for homogeneous assays.
Schroeder and Neagle describe a system that uses low angle laser scanning illumination and a mask to selectively excite fluorescence within approximately 200 microns of the bottoms of the wells in standard 96 well plates in order to reduce background when imaging cell monolayers. (J. Biomol. Scr. 1:75-80, 1996). This system uses a CCD camera to image the whole area of the plate bottom. Although this system measures signals originating from a cell monolayer at the bottom of the well, the signal measured is averaged over the area of the well and is therefore still considered a homogeneous measurement, since it is an average response of a population of cells. The image is analyzed to calculate the total fluorescence per well for cell-based homogeneous assays.
Proffitt et. al. (Cytometry 24:204-213, 1996) describe a semi-automated fluorescence digital imaging system for quantifying relative cell numbers in situ, where the cells have been pretreated with fluorescein diacetate (FDA). The system utilizes a variety of tissue culture plate formats, particularly 96-well microplates. The system consists of an epifluorescence inverted microscope with a motorized stage, video camera, image intensifier, and a microcomputer with a PC-Vision digitizer. TURBO PASCAL® software controls the stage and scans the plate taking multiple images per well. The software calculates total fluorescence per well, provides for daily calibration, and configures for a variety of tissue culture plate formats. Thresholding of digital images and the use of reagents that fluoresce only when taken up by living cells are used to reduce background fluorescence without removing excess fluorescent reagent.
A variety of methods have been developed to image fluorescent cells with a microscope and extract information about the spatial distribution and temporal changes occurring in these cells. A recent article describes many of these methods and their applications (Taylor et al., Am. Scientist 80:322-335, 1992). These methods have been designed and optimized for the preparation of small numbers of specimens for high spatial and temporal resolution imaging measurements of distribution, amount and biochemical environment of the fluorescent reporter molecules in the cells.
Treating cells with dyes and fluorescent reagents, imaging the cells, and engineering the cells to produce a fluorescent reporter molecule, such as modified green fluorescent protein (GFP), are useful detection methods (Wang et al., In Methods in Cell Biology, New York, Alan R. Liss, 29:1-12, 1989). The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has an excitation maximum at 395 nm, an emission maximum at 510 nm and does not require an exogenous factor. Uses of GFP for the study of gene expression and protein localization are discussed in Chalfie et al., Science 263:802-805, 1994. Some properties of wild-type GFP are disclosed by Morise et al. (Biochemistry 13:2656-2662, 1974), and Ward et al. (Photochem. Photobiol. 31:611-615, 1980). An article by Rizzuto et al. (Nature 358:325-327, 1992) discusses the use of wild-type GFP as a tool for visualizing subcellular organelles in cells. Kaether and Gerdes (FEBS Letters 369:267-271, 1995) report the visualization of protein transport along the secretory pathway using wild-type GFP. The expression of GFP in plant cells is discussed by Hu and Cheng (FEBS Letters 369:331-334, 1995), while GFP expression in Drosophila embryos is described by Davis et al. (Dev. Biology 170:726-729, 1995). U.S. Pat. No. 5,491,084, incorporated by reference herein, discloses expression of GFP from Aequorea victoria in cells as a reporter molecule fused to another protein of interest. Mutants of GFP have been prepared and used in several biological systems. (Hasselhoff et al., Proc. Natl. Acad. Sci. 94:2122-2127, 1997; Brejc et al., Proc. Natl. Acad. Sci. 94:2306-2311, 1997; Cheng et al., Nature Biotech. 14:606-609, 1996; Heim and Tsien, Curr. Biol. 6:178-192, 1996; Ehrig et al., FEBS Letters 367:163-166, 1995).
The ARRAYSCAN™ System, as developed by Cellomics, Inc. (U.S. Pat. No. 5,989,835) and U.S. application Ser. No. 09/031,271 filed Feb. 27, 1998; both incorporated by reference herein in their entirety) is an optical system for determining the distribution, environment, or activity of luminescently labeled reporter molecules on or in cells for the purpose of screening large numbers of compounds for specific biological activity. The ARRAYSCAN™ System involves providing cells containing luminescent reporter molecules in an array of locations and scanning numerous cells in each location, converting the optical information into digital data, and utilizing the digital data to determine the distribution, environment or activity of the luminescently labeled reporter molecules in the cells. The ARRAYSCAN™ System includes apparatus and computerized method for processing, displaying and storing the data, thus augmenting drug discovery by providing high content cell-based screening, as well as combined high throughput and high content cell-based screening, in a large microplate format.
Microfluidics
Efficient delivery of solutions to an array of cells attached to a solid substrate is facilitated by a microfluidic system. Methods and apparatus have been described for the precise handling of small liquid samples for ink delivery (U.S. Pat. Nos. 5,233,369; 5,486,855; 5,502,467), biosample aspiration (U.S. Pat. No. 4,982,739), reagent storage and delivery (U.S. Pat. No. 5,031,797), and partitioned microelectronic and fluidic device array for clinical diagnostics and chemical synthesis (U.S. Pat. No. 5,585,069). In addition, methods and apparatus have been described for the formation of microchannels in solid substrates that can be used to direct small liquid samples along the surface (U.S. Pat. Nos. 5,571,410; 5,500,071; 4,344,816,).
For purposes of integrated high throughput and high content cell based screening, particularly for live-cell imaging, an optimal microfluidic device would comprise a fluidic architecture that permits the closest possible well spacing (i.e.: highest possible well density), wherein the fluidic architecture is integrated with the cell array substrate to permit efficient fluid delivery to the cells, and eliminating the need for pipetting fluids in and out of wells. Such optimal microfluidic devices would be advantageous for cell arrays with sub-millimeter inter-well distances because it is unwieldy, if not impossible, to pipette fluids with such a high degree of spatial resolution and accuracy. Furthermore, such integrated devices could be directly used for cell based screening, without the need to remove the cell substrate from the fluidic architecture for imaging the cells.
An optimal microfluidic device for cell based screening might further comprise a closed chamber to permit environmental control of the cells, and preferably would not directly expose the cells to electro-kinetic forces, which may affect the physiology of the cells on the substrate. For example, electrohydrodynamic pumping is less effective with polar solvents (Marc Madou, Fundamentals of Microfabrication, CRC Press, Boca Raton, 1997, p. 433). Electro-osmosis is typically accompanied by some degree of electrophoretic separation of charged medium components, such as proteins.
U.S. Pat. No. 5,603,351 (‘the 351 patent’) describes a microfluidic device that uses a multilevel design consisting of two upper levels with channels and a bottom level with reaction wells. However, this device is not designed for use in cell based screening. The '351 patent does not disclose a substrate containing cells or cell binding sites. The disclosed microfluidic network is designed to allow two or more reagents to be combined in a reaction well, as opposed to an optimal cell screening microfluidic system that allows living cells cultured on the well bottoms to be exposed in serial fashion to two or more different fluids. The '351 patent discloses a device with the wells etched into the substrate at a maximal well density of 50 wells/inch2. Furthermore, the substrate must be detached from the fluidic array for incubation and/or analysis. Finally, the '351 patent discloses a system of electrically-controlled electrohydrodynamic valves within the matrix of the wells that are less effective with aqueous media used in cell culture, and also may limit the degree of close-spacing between wells in the array of wells.
U.S. Pat. No. 5,655,560 discloses a clog-free valving system, comprising a fluid distribution system with multiple inputs and multiple outputs incorporating a crossed array of microchannels connected vertically at crossing points by TEFLON® valves. However, this patent does not disclose a substrate containing a cell array, nor an integrated fluidic device in combination with the substrate, nor a well density that is optimal for cell-based screening.
U.S. Pat. No. 5,900,130 (the '130′ patent) describes the active, electronic control of fluid movement in an interconnected capillary structure. This patent does not teach a fluidic architecture that maximizes the area of the cell substrate that can be occupied by cell binding sites. Nor does this patent disclose a substrate containing a cell array, nor an integrated fluidic device in combination with the substrate. Furthermore, the patent only teaches the control of fluid flow by application of an electric field to the device.
U.S. Pat. No. 5,910,287 describes multi-well plastic plates for fluorescence measurements of biological and biochemical samples, including cells, limited to plates with greater than 864 wells. This patent does not describe a microfluidic device with a fluidic architecture integrated with the cell array substrate. Nor does the patent disclose a closed chamber to permit environmental control of the cells on the substrate.
Thus, none of these prior microfluidic devices provide a fluidic architecture that permits the closest possible well spacing (i.e.: highest possible well density), wherein the fluidic architecture is integrated with the cell array substrate to permit efficient fluid delivery to the cells, and thus eliminating the need for pipetting fluids in and out of wells. Furthermore, prior microfluidic devices that comprise an array of wells use electrically-controlled electrohydrodynamic valves within the matrix of the wells that would be less effective if used with aqueous media for cell culture, and which limit the well density.
While the above advances in cell array, optical cell physiology reading, and microfluidic technologies provide supporting technologies that can be applied to improved high throughput and high content cell-based screening, there remains a need in the art for integrated devices and methods that further decrease the amount of time necessary for such screening, as well as for devices and methods that further improve the ability to conduct high throughput and high content cell-based screening and the ability to flexibly and rapidly switch from one to the other. In particular, devices and methods that maximize the well density, thereby increasing the number of wells that can be imaged in at one time, and thus greatly increasing the throughput of a screen while maintaining adequate resolution of the image, would be very advantageous.
The drug discovery industry already uses 96- and 384-well microplates and is in transition towards the use of 1536-well plates. However, further increases in well density using prior technology are unlikely because of the great difficulty of pipetting liquids in and out of very small diameter wells.